This invention relates to methods and systems for NMR analysis of analytes in small volumes. More particularly, this invention relates to methods and systems employing NMR microcoils for analysis of analytes in, for example, nanoliter sample volumes.
Nuclear magnetic resonance spectroscopy, or NMR, is a powerful and commonly used method for analysis of the chemical structure of molecules. NMR provides spectral information as a function of the electronic environment of the molecule and is nondestructive to the sample. In addition, reaction rates, coupling constants, bond-lengths, and two- and three-dimensional structure can be obtained with this technique. NMR combined with liquid chromatography or capillary electrophoresis was demonstrated as early as 1978 using stopped flow (Watanabe et al. (1978) Proc. Jpn. Acad. 54:194), and in 1979 with continuous flow (Bayer et al. (1979) J. Chromatog. 186:497-507), though limitations due to solvent as well as inherent sensitivity curtailed the use of the method. That is, the inherent insensitivity of the NMR method limited its usefulness as a detection method for liquid phase analysis of very small samples, such as effluent from an analyte focusing or concentrating method (those two terms being used here interchangeably unless otherwise indicated), e.g., a liquid chromatography or capillary electrophoretic separation. Conventional NMR spectrometers typically use relatively large RF coils (mm to cm size) and samples in the mu.l to ml volume range, and significant performance advantages are achieved using NMR microcoils when examining very small samples. NMR microcoils are known to those skilled in the art and are shown, for example, in U.S. Pat. No. 5,654,636 to Sweedler et al., and in U.S. Pat. No. 5,684,401 to Peck et al., and in U.S. Pat. No. 6,097,188 to Sweedler et al., all three of which patents are incorporated herein by reference in their entireties for all purposes. A solenoid microcoil detection cell formed from a fused silica capillary wrapped with copper wire has been used for static measurements of sucrose, arginine and other simple compounds. Wu et al. (1994a), J. Am. Chem. Soc. 116:7929-7930; Olson et al. (1995), Science 270:1967-1970, Peck (1995) J. Magn. Reson. 108(B) 114-124. Coil diameter has been further reduced by the use of conventional micro-electronic techniques in which planar gold or aluminum R.F. coils having a diameter ranging from 10-200 .mu.m were etched in silicon dioxide using standard photolithography. Peck 1994 IEEE Trans Biomed Eng 41(7) 706-709, Stocker 1997 IEEE Trans Biomed Eng 44(11) 1122-1127, Magin 1997 IEEE Spectrum 34 51-61, which are also incorporated herein by reference in its entirety for all purposes.
Miniature total analysis systems (xcexc-TAS) are discussed in Integrating Microfluidic Systems And NMR Spectroscopyxe2x80x94Preliminary Results, Trumbull et al, Solid-State Sensor and Actuator Workshop, pp. 101-05 (1998), Magin 1997 IEEE Spectrum 34 51-61, and Trumbull 2000 47(1) 1-6 incorporated herein by reference in its entirety for all purposes. The Trumbull et al. device integrated multiple chemical processing steps and the means of analyzing their results on the same miniaturized system. Specifically, Trumbull et al. coupled chip-based capillary electrophoresis (CE) with nuclear magnetic resonance spectroscopy (NMR) in a xcexcL-TAS system. Linewidths of 1.4 Hz have been demonstrated using single turn planar NMR coils integrated with microfluidic channels.
Capillary-based liquid chromatography and microcoil NMR have compatible flow rates and sample volume requirements. Thus, for example, the combination of the Waters CapLC(trademark) available from Waters Corporation (Milford, Mass., USA) and the MRM CapNMR(trademark) flow probe available from MRM Corporation (Savoy, Ill.), a division of Protasis Corporation (Marlborough, Mass., USA) provides excellent separation capability in addition to UV-VIS and NMR detection for mass-limited samples. The Waters CapLC(trademark) has published flow rates from 0.02 xcexcL/minute to 40 xcexcL/minute. A typical CapLC on-column flow rate is 5 xcexcxcexcL/min, the autosampler-injected analyte volume is 0.1 xcexcL or more, and accurate flow rates are achieved through capillary of typically 50 xcexcm inner diameter. The NMR flow cell has a typical total volume of 5 xcexcL with a microcoil observe volume of 1 xcexcL. A typical injected sample amount for CapLC-xcexcNMR analysis is a few xcexcg (nmol) or less.
Conventional scale LC-NMR systems (flow rates about 1 ML/min) typically employ solvent gradients of 2% per minute to 4% per minute. The probes have typical flow cell volumes of about 150 xcexcL and typical observe volumes of 30-60 xcexcL. In such systems, the application of a solvent gradient followed by stopped flow can initially result in severely distorted NMR line shapes due to the magnetic inhomogeneity caused by nonuniform solvent composition in the flow cell. After a reasonable equilibration time, if the solvent gradient is no longer too steep, line shape is seen to recover from the initial stopped flow conditions. However, steep solvent gradients typically result inmagnetic distortions so severe that NMR is practically precluded due to the extremely long recovery time ( greater than  greater than 1 hr) necessary to achieve high spectral resolution.
Capillary scale systems also are known. In such systems, a capillary-based analyte extraction chamber can be connected to an NMR flow cell, such as by being positioned as an operation site along a capillary channel extending to the NMR flow cell. Exemplary such integrated capillary-based analyte extraction chambers are shown in U.S. Pat. No. 6,194,900, the entire disclosure of which is incorporated herein by reference for all purposes.
There is a need in the art for improved NMR methods for analyzing analytes. There is a particular need for improved NMR methods of analyzing small samples, especially samples of less than 10 xcexcL or even less than 1.0 xcexcL.
Additional objects, advantages and novel features of the invention will be set forth in part in the description which follows, and in part will become apparent to those skilled in the art upon examination of the following, or may be learned by practice of the invention.
Solvent gradients play an important role in NMR analysis, as indicated above. In accordance with the methods disclosed here, a flow of analyte sample fed to an NMR flow cell has a mobile phase with advantageously steep solvent gradient.
In accordance with a first aspect, a method of analyzing an analyte comprises feeding a fluid flow of analyte sample fluid, that is a fluid containing or suspected of possibly containing one or more analytes of interest in a multi-component mobile phase to a fluid channel of a nuclear magnetic resonance (NMR) probe. The analyte sample fluid is fed to an NMR flow cell in the fluid channel. The flow cell comprises an RF microcoil operably associated with an enlarged containment region. The mobile phase of the analyte sample flowing to the NMR flow cell has a solvent gradient greater than 10% per minute. In certain preferred embodiments, the mobile phase of the analyte sample fluid has a solvent gradient of 10%-30% per minute or more. Coupled with NMR detection, this variation in (mobile phase) solvent composition is fed directly into the NMR flow cell. Exemplary mobile phases include aqueous/organic mobile phases, and any others suitable to the particular application, of which many are known to those skilled in the art. It will be recognized that the methods and systems disclosed here provide an advantageous technological advance. An operator of a liquid chromatography system or other analyte extraction means generally wants full access and control over the solvent composition of the analyte sample flow, as this chemistry directly influences the effectiveness of the separation. In many instances, the compositional mixture of two or more solvents will be intentionally varied to provide a desired effect. The necessary or useful rate of solvent compositional change is in some instances greater than 10% per minute, and the methods and systems disclosed here render such steep solvent gradients practical.
In accordance with another aspect, methods as disclosed immediately above further comprise holding a volume of the analyte sample fluid in the NMR flow cell for an equilibration time of less than 1 hour, preferably less than 30 minutes, e.g., 10 minutes, and then actuating NMR analysis of analyte in an observe volume of the NMR flow cell. Preferably the observe volume of the sample fluid in the NMR flow cell is less than 5 xcexcL, e.g., about 1 xcexcL. The sample volume may be held in the NMR flow cell, for example, by permanently or temporarily stopping the flow of analyte sample fluid in the NMR probe. In capillary-based embodiments of the systems and methods disclosed here, the analyte sample fluid has a fluid flow rate typically less than about 5 xcexcL/minute. In substrate-based, i.e., micro-scale, embodiments of the systems and methods disclosed here, the analyte sample fluid has a fluid flow rate typically less than 1 xcexcL/minute.
In accordance with another aspect, methods as disclosed immediately above further comprise processing sample fluid containing (here and in the appended claims meaning actually containing or suspected of possibly containing) an analyte in an analyte extraction device to produce analyte sample fluid that is then (optionally with one or more intervening processing steps) fed to the NMR flow cell comprising an RF microcoil. Exemplary analyte extraction devices include liquid chromatography (LC) devices, capillary electrophoretic and/or capillary electrochromatographic (CE/CEC) devices, electric field gradient focusing (EFGF, Koegler 1996 J. Chromatography 229 229-236, Koegler 1996 Biotech Prog 12(6) 822-836) and dynamic field gradient focusing (DFGF, Huang 1999 Anal. Chem. 71(8) 1628-1632) devices and the like. In certain preferred embodiments, sample fluid is processed in a capillary-based, i.e., a capillary scale, analyte extraction device, to produce analyte sample fluid that is then (optionally with one or more intervening processing steps) fed to a capillary NMR flow cell comprising an RF microcoil. Exemplary capillary scale analyte extraction devices include capillary LC and CE devices, e.g., the Waters CapLC(trademark) column. A steep solvent gradient is then used to separate and remove analyte (e.g., an organic/water mobile phase with a 10-30% per minute gradient). The one or more analyte peaks are then individually stopped in the capillary NMR flow cell to acquire high resolution NMR data, e.g., a proton spectrum, after an equilibration time of typically less than 30 minutes. This capillary approach enables preconcentration of analytes on a separation column from relatively large volumes, having initial concentrations of typically less than 1 mM, and good separation efficiency coupled with good NMR spectral resolution. In particular, to make NMR practical as a means of detection for analytical separations it is highly advantageous that steep solvent gradients are used in conjunction with short equilibration times and yet good NMR spectral resolution is obtained.
In accordance with certain preferred embodiments, methods as disclosed above employ analyte sample volumes less than about 10 microliters. Exemplary such embodiments dispose analyte sample in an NMR flow cell wherein the inside dimension of the associated microcoil is less than about 1 mm. A static magnetic field is generated about the analyte sample using a magnet well known to those skilled in the art. Using NMR techniques well known to those skilled in the art, the free induction decay signal from the analyte is received by the NMR microcoil and preferably has a spectral linewidth of better than 0.1 parts per million (ppm), more preferably better than 0.01 ppm, e.g. 0.001 ppm.
In certain preferred embodiments, a miniaturized analysis system is employed for liquid phase sample analysis. Such methods and systems, referred to in some instances here and in the appended claims as substrate-based methods and systems, employ: a microfabricated support body or manifold in the form of a cylinder, chip, laminated planar substrate or the like, having in or on the manifold one or more straight or branched microfabricated microchannels; optionally a housing, e.g., a cover plate, arranged over the manifold, optionally cooperating with the manifold to form sample processing channels or compartments; an inlet port for feeding fluid from an external source into the NMR probe; and an NMR flow cell comprising an NMR RF microcoil, in fluid communication with the inlet via one or more of the microfabricated microchannel. As used here, the terms xe2x80x9cmicro-scalexe2x80x9d and xe2x80x9cmicrofluidicxe2x80x9d means the manifold operates effectively on micro-scale fluid samples, typically having volumes less than about 1 uL (i.e., 1 microliter), e.g., about about 0.1 microliter to 1.0 microliter, and fluid flow rates less than about 1 uL/min, for example 100 nanoliters/min. As used herein, the term xe2x80x9cmicroscalexe2x80x9d also refers to flow passages or channels and other structural elements of a substrate, e.g., a multi-layer laminated substrate. For example, 1 or more microchannels of the substrate preferably have a cross-sectional dimension (diameter, width or height) between 500 microns and 100 nanometers. Thus, at the small end of that range, the microchannel has cross-sectional area of about 0.01 square microns. Such microchannels within the laminated substrate, and chambers and other structures within the laminated substrate, when viewed in cross-section, may be triangular, ellipsoidal, square, rectangular, circular or any other shape, with at least one and preferably all of the cross-sectional dimensions transverse to the path of fluid flow is microscale. It should be recognized, that one or more layers of a laminated substrate may in certain embodiments have operative features, such as fluid channels, reaction chambers or zones, accumulation sites etc. that are larger than microscale. The substrates disclosed here provide effective microcoil NMR devices and systems with good speed of analysis, decreased sample and solvent consumption, increased detection efficiency, and in certain embodiments disposable fluid-handling devices.
The capillary-scale and micro-scale embodiments of the NMR methods and systems disclosed here provide significant commercial advantage over conventional (larger scale, e.g. larger than capillary) NMR systems including, for example: less sample fluid is required, which in certain applications can present significant cost reductions, both in reducing product usage (for example, if the test sample is a biological sample or is taken from a product stream) and in reducing the waste stream disposal volume. In addition, they can, in accordance with preferred embodiments, be produced employing MEMS and other known techniques suitable for cost effective manufacture of miniature high precision devices.
The methods disclosed here are carried out, in certain preferred embodiments, in a system embodied in an NMR probe comprising multiple processing sites (i.e., stages or devices operative to carry out fluid operations on a sample) integrated together in a common NMR manifold, such as capillary-based methods and systems or substrate-based methods and systems with fluid communication between the processing sites being provided by microchannels and the like defined by the manifold, i.e., formed in or on the manifold. The multiple processing sites in such systems and methods include at least an analyte extraction chamber and an NMR flow cell downstream of the analyte extraction chamber, i.e., positioned to receive analyte sample fluid from the analyte extraction chamber. For example, a biological sample, synthetic intermediatary, metabolite, contaminant, natural product or the like is fed directly to the NMR probe. The sample is then prepared as required to produce analyte sample fluid, e.g., filtration, solid phase extraction, capillary electrophoresis, liquid chromatography, etc. The prepared analyte sample fluid is then passed to an NMR flow cell for NMR detection. The NMR detection chamber has an integrated NMR radio frequency microcoil embedded or otherwise integrated directly in the manifold. Following detection, the sample can be discarded or, optionally, stored in the NMR manifold indefinitely, either in the NMR flow cell or other chamber defined by the manifold. Optionally, the sample is transported via capillary or on-chip to a further analytical station or storage site. In certain preferred embodiments the total analysis requires less than about 1 .mu.L of sample.